Electrochemical testing system and method

ABSTRACT

An electrochemical measuring system which effects, measures and sorts charge transfer reactions of selected substances in a sample solution is provided. The system includes a novel electrode which is adapted to measure simultaneously on at least two electrodes at least two electrolytic potentials with reference to a reference potential. In a preferred form the electrode comprises a hollow, cylindrical body formed of an electrically insulating material. The electrode is open at least at one end and has a generally smooth cylindrical inner surface. A plurality of electrically discrete active electrode segments are mounted on the inner surface of the electrode with their active surfaces substantially flush with the generally smooth cylindrical inner surface. Completing the electrochemical measuring system are a stirring means for creating a relatively high degree of mixing adjacent the electrode active surfaces, means for charging liquid samples to the cell, and means connecting at least two of the electrically discrete active electrode segments to different electrical potentials. In use a sample to be tested is charged to the cell, and stirring is commenced. One of the electrically discrete active electrode segments is held at a potential at which a selected substance of interest and also one or more interferring substances responds, while another of the electrode segments is held at a potential at which only the interferring substances respond. The presence of a substance of interest can be determined by subtracting the signals from the one and another electrode segments, and its quantity determined by integrating the signal difference.

The present application is a continuation-in-part of our copendingapplication Ser. No. 868,654, filed Jan. 11, 1978 now abandoned.

Various electrochemical systems are known in the art for detecting thepresence of and/or measuring the concentration of various substances ofinterest in sample solutions suspected of containing the selectedsubstances, and find utility in a variety of environmental, medical andindustrial applications. Generally, such systems are employed inanalyzing for metallic ions of interest, although systems also exist forthe detection of non-metals such as cyanide ion, sulfur dioxide andhalogen, and for certain organic materials.

One type of prior art electrochemical analysis employs gravimetricmethods in which a deposit formed by electrical action is weighed on ananalytical balance. Gravimetric methods are prone to weighing errors,require a skilled technician, and are relatively time consuming andinsensitive.

Another type of prior art electrochemical analysis employs ion-selectiveelectrodes. A number of ion-selective electrodes have been devised fortesting for a variety of ions of interest and are considered to bereliable and relatively easy to use. However, a number of substances ofinterest in the environmental industrial and medical fields cannot bemeasured with ion selective electrodes. Moreover, ion-selectiveelectrodes respond logarithmically and thus generally are notsufficiently sensitive for measuring concentrations below about 10⁻⁵ to10⁻⁶ molar.

Polarographic analysis based on current voltage curves obtained withhanging drop mercury electrodes offers an advantage over ion-selectiveelectrodes of sensitivity in dilute solutions. A feature and requirementof classic hanging drop mercury polarographic electrolysis cells is thedropping mercury electrode, i.e., mercury droplets being dischargedperiodically into a solution from a fine bore capillary under a drivinghead of mercury. However, this very feature, which has permitted theinitiation of extremely useful polarographic methods in research work,mitigates against a more general use of classic polarographicelectrolysis cells as common analytical systems, and in particular astools for monitoring and controlling industrial process streams or forfield use testing in medical and environmental applications. Moreover,the characteristic periodic growth and fall of the mercury dropletscause oscillations in the current-voltage curves obtained using suchcells and thus prevent the establishment of standard curves. Otherproblems of hanging drop mercury electrodes which have essentiallylimited cells employing same to laboratory and experimental use includecondensor current build-up whenever a new mercury droplet is beingformed at the capillary, and limited surface area of the droplets whichlimits sensitivity of the electrode. In addition, formation of the tinymercury droplets is a delicate process which may be affected by a numberof incidental factors, including mechanical vibration, slant ofcapillary, and pulsation of test solutions into the capillary inletbetween drops. In this connection it should be noted that thereproducibility of droplets with regard to their drop line and mass ofmercury per drop must be practically perfect at all times to permitproper evaluation of the polarogram.

Still another type of prior art electrochemical measuring system is atechnique called coulometric shipping voltammetry. Coulometric strippingvoltammetry is a two-step process comprising electrodepositing theelectroactive material of interest on or in an indicating or workingelectrode and then electrodissolving or stripping the deposited materialback into solution. In anodic stripping voltammetry, the material to bemeasured is plated onto an electrode by applying a negative potentialover an extended time period, and then stripping the material off theelectrode over a relatively short period by sweeping to a positivepotential. The order of potential at which the elements of the materialare stripped off the electrode provides a qualitative analysis of thematerial, and the quantity of the current provides a quantitativeanalysis. Anodic stripping voltammetry offers the advantages of enhancedsensitivity, resolution, and reproducibility compared to classicalpolarographic analysis obtained using hanging drop mercury electrodes.By way of example, thin-film mercury/graphite composite electrodes havebeen employed in anodic stripping voltammetry systems for analyzing formetals at the sub-nanogram level. See, for example, the reported work ofWayne R. Matson, Reginald M. Griffin, and George B. Schreiber in "RapidSub-Nanogram Simultaneous Analysis of Zn, Cd, Pb, Cu, Bi and Ti", TraceSubstances in Environmental Health, University of Missouri, Dr. D.Hemphill, Ed; pp. 396-406, (1971). While electrochemically analyzingsolutions employing composite mercury/graphite electrodes by anodicstripping voltammetry, e.g. as taught by Matson et al, supra, mayprovide sub-nanogram sensitivity, the ability to rapidly and reliablydifferentiate and measure selected substances at the picogram level isnot generally possible using existing electrochemical measuringtechniques. Also, many metals interact with the electrode to form analloy of analgam. Thus, anodic and cathodic stripping voltammetry arelimited to detection of a relatively small number of species of metalsand non-metals. Obviously, the ability to operate at such lowconcentrations and on a wider variety of species would have majorcommercial utility in environmental, medical and industrialapplications.

It is thus a primary object of the present invention to provide a noveland improved system, i.e. method and apparatus, which overcomes theaforesaid and other problems and limitations of the prior art.

Another primary object is to provide a novel and improved method andapparatus for electrochemically analyzing a sample in order toqualitatively and/or quantitatively determine the presence of selectedsubstances in the sample.

Another object of the present invention is to provide an electrochemicalmeasuring system of the aforesaid type which is capable of rapidly andreliably operating at the picogram level of sensitivity.

A more specific object is to provide a novel and improved electrode foruse in electrochemical systems.

In order to effect the foregoing and other objects there is provided anelectrochemical measuring system which effects, measures and sortscharge transfer reactions of selected substances in a sample solution.The system includes a novel electrode which is adapted to measuresimultaneously on at least two electrodes at two electrolytic potentialswith reference to a reference potential. In a preferred form theelectrode comprises a hollow, cylindrical body formed of an electricallyinsulating material. The electrode is open at least at one end and has agenerally smooth cylindrical inner surface. A plurality of electricallydiscrete active electrode segments are mounted on the inner surface ofthe electrode with their active surfaces substantially flush with thegenerally smooth cylindrical inner surface. Completing theelectrochemical measuring system are a stirring means for creating arelatively high degree of mixing adjacent the electrode active surfaces,means for charging liquid samples to the cell, and means connecting atleast two of the electrically discrete active electrode segments todifferent electrical potentials. In use a sample to be tested is chargedto the cell, and stirring is commenced. One of the electrically discreteactive electrode segments is held at a potential at which a selectedsubstance of interest and also one or more interferring substancesresponds, while another of the electrode segments is held at a potentialat which only the interferring substances respond. The presence of asubstance of interest can be determined by subtracting the signals fromthe one and another electrode segments, and its quantity determined byintegrating the signal difference.

Yet other objects of the invention will in part appear obvious and willin part appear hereinafter. The invention accordingly comprises theapparatus possessing the construction, combination of elements, andarrangement of parts, and the process comprising the several steps andthe relation of one or more of such steps with respect to each of theothers, all of which are exemplified in the following detaileddescription, and the scope of the application as will be indicated inthe claims.

For a fuller understanding of the nature and objects of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings wherein:

FIG. 1 is a front view in perspective of a preferred form ofelectrochemical measuring apparatus according to the invention;

FIG. 2 is a front view, partially in section, or a preferred form ofelectrolytic cell of the apparatus of FIG. 1;

FIG. 3 is an end view, in cross-section of the sample solution stirringmember of apparatus of FIG. 1;

FIG. 4 is a fragmentary plan view, in cross-section of a preferred formof sensing electrode element of the apparatus of FIG. 1;

FIG. 5 is a diagrammatic view of a flow detector element of theapparatus of FIG. 1;

FIG. 6 is a block diagram of the electrical and pneumatic controls andfunctions of the apparatus of FIG. 1;

FIG. 7 is a plot showing the current in microamperes, μA, verses thepotential in volts verses a standard silver/silver halide referenceelectrode, obtained in accordance with the present invention; and

FIGS. 8 and 9 are front views, in perspective, of alternative forms ofelectrolytic cells in accordance with the present invention.

The present invention is based on measurements of electrochemicalreactions of selected substances in solution under controlled potentialconditions. As is well known in the art, when electroactive substancesare dissolved in a solvent to form a reagent of electrolyte, and anelectrical current passed through the electrolyte between an anode and acathode disposed therein, positive ions will be attracted to thenegatively charged cathode where their charge will be neutralized, whilenegative ions will move towards and be discharged at the anode. Theelectrical potentials at which such electrochemical reactions occur willvary depending upon the particular substances involved. By way ofexample, consider an aqueous solution which contains both iron andcopper ions. Iron normally exhibits a valence of two or three, whilecopper normally exhibits a valance of one or two. The electricalpotential at which ferric ions (Fe⁺³) in solution may be reduced toferrous ions (Fe⁺²) is a constant at a given temperature. Likewise, theelectrical potential at which cupric ions (Cu⁺²) in solution may bereduced to cuprous ions (Cu⁺¹) is also a constant at a giventemperature, and is different from the electrical potential at which thereduction of ferric ions to ferrous ions occurs. (The electricalpotential at which such reactions occur are approximately described bytables of standard or formal potentials). The absolute value of theelectrical potential of ions of solution is indeterminate. However,electrochemical reactions for a particular species are described interms of a potential versus a standard reference couple such as H₂ H⁺.The magnitude of the potential is a measure of the potential that has tobe applied versus a standard reference electrode to force chargetransfer to occur. The electrical potential at which such reactions willoccur is referred to as the "Charge transfer potential".

Assigning an arbitrary value of zero to hydrogen, the potential E of anelectrochemical reaction may thus be written according to the followingreaction: ##EQU1## where n is the number of Faradays, A_(P) and A_(R)are activities of the product reactants, and x and y are correspondingcoefficients of the electrochemical reactions. Thus, the potential E_(o)is the standard potential related to the particular reaction. E is apotential applied to drive the reaction either to reactants or productsaccording to the equilibrium condition described by equation (1). Underconditions where the E applied is large enough to drive the reaction tovirtual completion at equilibrium, the current derived will beproportional to the concentration of the reactant in the solution.However, background noise prevents direct measurement of most samplesolutions and in the case of very dilute solutions may prevent directmeasurement in many instances. (As used herein the term "backgroundnoise" is intended to refer both to major interference factors such asthe presence in the solution of other electroactive materials which, byvirtue of their electrical activity in the solution, respond to the sameelectrical potential as the ion of interest, and also major non-Faradaicinterference factors such as capacitance signals of the electrode in thesolution due to the existence of a boundary layer of still solutionadjacent the active surfaces of the electrode, bulk solution signals,inherent Faradaic signals, electrode settling signals and the like). Afeature and advantage of the present invention resides in theelimination of and/or cancellation of background noise through acombination of electrochemical manipulations and electrode geometry.

Further understanding of the features and advantages of the presentinvention will be had from the following detailed description of onepreferred embodiment of the invention which illustrates anelectrochemical testing system for measuring the iron content in serumor blood. It will be understood however, that the system of the presentinvention may be advantageously employed for detecting the presence ofand measuring the concentration of various other substances of interestin sample solution.

Referring to FIG. 1, there is illustrated an electrochemical measuringapparatus indicated generally at 10 including a base 11. Mounted on base11 by means of upright support 12 is a cabinet 13 whose front face actsas a control panel 14. Mounted on the panel are various control meansincluding a a display panel 16, function buttons including a standbybutton 17, an "autoblank" control button 18, an "autoblank set" button19, and a calibration knob 20. Also positioned on the control panel 14is an off-on button 22, a flow indicator 23 suitably labelled to showthat a prior sample is flushed out and a new test may be started. Alsoon the control panel are a start button 24, and a "running" indicator25. For convenience it is preferred that the controls be combinations ofpush-buttons and indicating lights, and in the actual apparatus suchcombination buttons and lights are used.

Depending from the bottom of cabinet 13 is a cell assembly 27 indicatedin outline and shown in further detail in FIG. 2. Positioned on base 11are two containers 28 and 29, suitably connected by plastic tubing orthe like to the cell block. Container 28 receives flushed cell contentsat the end of each run and container 29 holds a supply of fresh cellliquid or electrolyte.

FIG. 2 shows the cell assembly of the apparatus of FIG. 1 comprisinggenerally a cell block 33 and a sensing electrode 34 mounted therein.Cell block 33 comprises a suitable mounting piece such as, for example,a plastic block having a screw threads 35 or other mounting means at theupper end. A vertical channel or cylindrical hollow 36 runs through thecell block and communicates with the interior of sensing electrode 34.Two passageways, the first an inlet passage 38 to receive a sample to betested which may, for example, be by means of a pipette (not shown)inserted into channel 38, and the second an outlet passageway 39 forcell liquid. Cell block 33 is formed of a liquid-impervious, rigid,electrically insulating, chemically inert material such as unplasticisedpolyvinyl chloride, polytetrafluoroethylene fluorocarbon resins or thelike.

The bottom of channel 36 is recessed to receive sensing electrode 34.Sensing electrode 34 is in the form of a hollow cylinder, and the innersurface of the electrode and the inner surface of channel 36 are flushand as smooth as possible so as to minimize the material caughttherebetween. In actual practice the electrode is permanently mounted inthe cell block by suitable means such as, for example, by an epoxy resinor the like, and the inner surface of the joint between the two ismachined smooth.

At the bottom of sensing electrode 34 is a seal and connector device 46which may, for example, be in the form of a plastic plug molded to thesensing electrode 34 having a screw thread connection 35a for connectinga pipe or hose thereto and having a channel 49 extending therethrough. Acontinuous passage is thus formed, and electrolyte or other contents ofthe cell can be flushed out by passing fresh electrolyte or other liquidin through channel 40 and out through outlet 39 in the cell block abovethe electrode.

As mentioned supra an important feature and advantage of the presentinvention is the ability to differentiate between electrical signalsrepresentative of the charge transfer electrolytic reaction of selectedsubstances of interest, and electrical signals derived from the bulksample solution, interferring substances and other background noise.This feature and advantage is made possible in part by the constructionof sensing electrode 34. Sensing electrode 34 comprises a generallycylindrical body of block epoxy having mounted therein a plurality ofactive electrode segments. The electrode body comprises an electricallyinsulating material such as a polymeric material while the activeelectrode segments comprise a suitable electrode base such as graphite,pyrolytic graphite or platinum, or the active electrode segments maycomprise coatings of active electrode material such as mercury or gold.In practice, at least two electrically discrete electrode areas areemployed, for example, in the form of rings or bands of active electrodesurface on the inside of a hollow electrode body. The segments areseparated by electrically insulating bands on the inside of theelectrode. Such an electrode can be formed by holding segments of activeelectrode material in the form of rings in desired position and moldingthe rings with an electrically insulating such as an epoxy resin to forma cylinder. Sensing electrode 34 is shown in further detail in FIG. 4.For convenience of illustration sensing electrode 34 has been shown ascomprising two active testing electrode segments, a counter or powersupplying electrode segment, and a reference electrode segment asfollows:--a first active testing electrode segment 42, a second activetesting electrode segment 43, a third counter electrode segment 44, anda reference electrode segment 45. The first, second and third activeelectrode segments 42, 43 and 44 are formed of suitable electrodematerial such as graphite or the like while the reference electrodesegment 45 is formed of silver, palladium or the like. Electrodesegments 42, 43, 44 and 45 each comprise a cylindrical ring embeddedinto a cylindrical electrode body 41. The electrode segments are spacedapart by a narrow gap so as to be electrically insulated one from theother, and the electrode segments are mounted so that the activesurfaces are substantially flush with the inner surface of electrodebody 41 so that the inner surface of the entire electrode 34 is a smoothas possible. Electrical connections (not shown in FIG. 4) are providedto each of the electrode segments and are suitably connected to theapparatus by means of a four wire lead terminating in a four-prongedplug as shown in FIG. 2. Obviously the electrode may comprise additionalactive electrode segments.

Positioned within electrode 34 is a stirring means 50. Stirring means 50is mounted for rotation within the electrode body by means of rod 51.Stirring means 50 and rod 51 are formed of electrically insulating andchemically inert materials such as molded resin. The lower end ofstirring means 50 is slightly wedge-shaped or cone-shaped, and isgenerally close fitting within the electrode body. A diagonal groove 52which is better seen in FIG. 3 runs along the surface of the stirringmeans 50. When rotated in the direction shown by arrow 54, groove 52creates a high degree of mixing or turbulence closely adjacent to theactive surfaces of electrode segments 42, 43, 44 and 45 so as tominimize the thickness of the boundary layer of still solution adjacentthe active surfaces of the electrode segments, while maximizing masstransfer to the electrode surfaces.

In use off-on button 22 is first activated. Ordinarily, the apparatuswill be left running in a standby condition overnight and will be turnedoff if it is to be left idle for a period of a week or more. At thestart of each week, or for purposes of abundant caution at the start ofeach day, the apparatus may be calibrated. It is first operated with thecalibration button in operating position to standardize the electronicsas will be hereinafter described. A blank sample of reagent is runfirst. Then the "auto-blank" button 19 is set, holding the calibration.Next a standard sample of known ion concentration is introduced into thecell 27 and the apparatus run through a cycle. When it has been properlystandardized, the calibration knob 20 is adjusted so that the reading inthe display panel 16 corresponds with the known ion quantity in thestandard calibration sample.

A plastic tube or pipe 40 (not shown in FIG. 2) connects the cellassembly 27 to the apparatus. At a selected point along tube 40 andpreferably within cabinet 13 is a flow detector illustrateddiagrammatically in FIG. 5. An emitter 55 or other light source ispositioned near a window at a point along tube 40. The window may be atransparent insert or the tube itself may be transparent. Oppositeemitter 55, i.e. on the opposite side of the tube 40 is a detector 56positioned adjacent a similar window. When tube 40 is empty or filledwith a gas the beam of light 57 from the emitter is quite diffuse. Whentube 40 is filled with a liquid such as the cell electrolyte flowingthrough the tube, the liquid acts as a lens and increases the sharpnessof focus of light beam 57. Detector 56 is adjusted for a threshold suchthat it can determine the presence of liquid in tube 40 and the lengthof time such liquid is present. The signal from detector 56 is employedto indicate that there has been flow of liquid through tube 40 for asufficient time to accomplish flushing out of cell electrolyte after asingle run so as to remove the sample therefrom.

In repetitive runs the cell 27 is repeatedly filled with an electrolyteand the cell stirring apparatus is constantly in operation to keep thecell contents uniform and mixed. A known quantity of a test sample isthen pipetted into a cell 27. The running indicator 25 lights to showthat the test is in operation. In a preferred embodiment of the presentinvention the display panel is a digital display which counts to zeroand then up to the number of micrograms per 100 milliliters of serum (μg%). When the digital display stops counting the test is complete. Aftera timed waiting period the cell electrolyte containing the sample isflushed into container 28 and a new supply of electrolyte is introducedinto the cell from container 29. When the start test indicator 24 lightsup again, the apparatus is ready for a next sample.

In FIG. 6 is shown a block diagram of electrical and fluid flow controlsfor the foregoing apparatus. A cell 27 such as the cell of FIG. 1 isconnected to have a reagent or electrolyte conveyed therethrough isindividual analysis quantities. A pump 60 pumps air through a line 61from a reagent container 62. A reagent valve 63 controls flow of thereagent to cell 27. Referring to FIG. 2, the reagent flows into lowerchannel 49 and thus into and through the cell 27. Another fluid line 65is positioned to carry the reagent or other liquid from the cell 27 pastan optical sensor 68 such as, for example, the sensor shown in FIG. 5.Line 65 then conveys the liquid to a drain container 69. A vacuum line70 returns to pump 60. Thus the flow of the liquid through cell 27 isinto the bottom of the cell and out through outlet 39 positioned abovethe cell. Preferably inlet channel 38 in cell 27 will be locatedslightly above outlet channel 39 so that liquid normally will flow outchannel 39 rather than channel 38.

For analyzing a sample the sample is dissolved in a solvent to form anelectrolyte or reagent. By way of example, for testing for iron in bloodor serum a small sample of blood or serum, typically a 5 to 100microliter sample is added to an electrolyte or chemical reagent whichreleases iron from its serum bonding and separates the transferpotentials of iron and its most usual interferring element, copper.

If total iron-binding capacity is being measured, the serum is firstfully saturated with iron, as by mixing it with an iron-containing ionexchange resin.

Preferably, the electrolyte or reagent for treating serum to releaseiron for testing comprises strong hydrochloric acid, e.g. between about51/2 and about 81/2 Formal, and preferably about 7 Formal, in a loweralcohol such as propanol or isopropanol. Methanol and ethanol have beenfound nearly as effective as propanol or isopropanol, but have thedisadvantage that they are more expensive, and they are more volatileand therefore more difficult to handle. Higher alcohols such as butanoland the like are operable, but are less compatible with stronghydrochloric acid. Other materials such as acetonitrile and acetone arealso operable but are less satisfactory partly because of lesssatisfactory performance and partly because of cost, volatility,toxicity and the like. The apparatus is calibrated in accordance withthe selected strengths of the hydrochloric acid.

The use of strong hydrochloric acid in lower alcohol as an electrolyteor reagent to release iron from its serum bonding and to separate thetransfer potential of iron and its most usual interferring substance,i.e. copper, in order to prepare the blood or serum forelectromechanical analysis for iron is believed novel.

In place of hydrochloric acid there may be employed other compoundshaving a high chlorine or halogen content, but such other compounds havenot been found to be fully satisfactory. For example, lithium chlorideis a more expensive source of chloride iron and also tends toprecipitate at least a portion of the serum. Hydrogen bromide is anothersource of halogen ion but is also more expensive and is notably moredifficult to work with and is corrosive.

Included in the reagent or electrolyte is an extremely minute quantityof silver ion in the range of about 200 parts per million which assistsin the operation of the silver reference electrode segment 45. Thereference potential is the silver ion potential, maintained by referenceelectrode segment 45. Accordingly, the reagent or electrolyte preferablywill include 7 Formal HCl in propanol together with 200 parts permillion silver ion and such reagent or electrolyte will release ironfrom serum or its iron binding components to make the iron available toelectromechanical measurement and also will permit separation of thecharge transfer potentials of iron and copper, and give reproducibleresults in the analysis of serum iron by electrochemical measurementtechniques in microliter sample quantities.

The prepared sample comprising a serum to be tested, together with ameasured quantity of a reagent or electrolyte is charged to cellassembly 27, and stirring commenced.

A potential control 70 applies two different electrical potentials 72and 73 to two of the active testing electrode segments, e.g. electrodesegments 42 and 43. Electrical potential 72 is set at a value whichcauses an electrochemical reaction of both iron and copper, whileelectrical potential 73 is set at a value which causes electrochemicalreaction of copper alone, as will be described in detail hereinafter. Areference potential is applied to the silver electrode segment 45, andanother potential is applied to counter electrode segment 44 andprovides a source of current to the cell. Alternatively counterelectrode segment 44 may be held at ground. The current or signals fromfirst and second testing electrode segments 42 and 43 are fed to a logicmodule which subtracts the first signal from the second and, if desired,applies a multiple for calibration purposes. By way of example, thecurrent or signals from the two active electrodes segements in the cell27 can be fed to a current convertor subtractor 75 with two variablegains for adjustment. The signal then goes to a signal accumulator 76,and then to a calibration blanking circuit 77 which also has a variablegain or calibrator 78. The signal from the calibration blanking circuit77 then is fed to a readout 80 and, in turn, to an autoblank control 81.The signal from the autoblank control is returned to the calibrationblanking circuit 77. When the calibration is correct, an autoblank set82 is operable to fix the circuits.

The electrochemical reactions which take place and are measured by theapparatus are the reduction of ferric ion to ferrous ion, the oxidationof ferrous ion to ferric ion, and the reduction of cupric ion to cuprousion. (Generally, material will not be deposited on the active electrodesegments, and accordingly these reactions may be considered to be"charge transfer" rather than electrolytic or electrodepositionreactions). At active electrode segment 42 there occurs the reduction offerric ion (Fe⁺³) to ferrous ion (Fe⁺²) and the reduction of cupric ion(Cu⁺²) to cuprous ion (Cu⁺¹). At active electrode segment 43 thereoccurs the reduction of cupric ion to cuprous ion and the oxidation offerrous ion to ferric ion. As a matter of choice, active electrodesegment 42 is set at the higher potential. The signal at the oneelectrode is subtracted from the other with the following result:

    (A) Fe.sup.+3 +e.sup.- →Fe.sup.+2 ; Cu.sup.+2 +e.sup.- →Cu.sup.+1 and                                     (2)

    (B) Fe.sup.+2 →Fe.sup.+3 +e; Cu.sup.+2 →Cu.sup.+1 -e. (3)

by subtraction

    (A)-(b)=Fe.sup.+3 and Fe.sup.+2 ; Cu→0.             (4)

As can be seen, the reduction of cupric to cuprous ion is cancelled outin the logic with the result that the total of iron content is thesignal which is fed to the digital or other readout.

Generally, the potential on active electrode segment 42 may be variedbetween about 0 to 1 volt while the potential on active electrodesegment 43 may be varied between about 0 to 300 millivolts from that ofsegment 42. For testing the serum iron in accordance with the foregoingtechnique active electrode segment 42 will be set at a potential ofabout 460 millivolts while active electrode segment 43 will be set at apotential of about 250 millivolts.

It is to be appreciated that the invention is not limited to themeasurement of serum iron, but that any electroactive substance may bedetected and measured using the foregoing process and apparatus. By wayof example, the electrochemical measuring system of the presentinvention may be used for detecting and measuring heavy metals such aszinc, cadmium, lead, copper, bismuth, gold, silver and thallium in bloodsamples. As is well known in the art such heavy metals normally arecomplexed with blood, and thus must be released before than can bemeasured. A number of reagents are known in the art and are availablecommercially for releasing such heavy metals from human blood. One suchreagent is called METEXCHANGE® and is available from EnvironmentalSciences Associates, Inc., of Bedford, Mass. The manufacturer describesthis reagent as comprising a dilute aqueous solution of calciumchloride, chromium tri-chloride, hydrogen ion, phosphate ion, acetateion and a dispersing agent. The mixture of calcium ion and chromium ionis said to cause release of complexed heavy metal in blood so that thetotal concentration of heavy metal can be effectively measured.

Moreover, the invention is not limited to the detection and measurementof heavy metals in biological samples. For example, heavy metalscomplexed with gasoline can be detected and measured in accordance withthe foregoing by dissolving gasoline samples in a reagent whichcomprises a dilute mixture of ICl, NaCl, N₂ H₄ HCl and a polyalcohol.The same reagent can be used to release various other heavy metals froma wide variety of organic samples. Other reagents which contain a metalion which will displace the heavy metal of interest from the complex canalso be used.

Additionally, a large number of organic substances are electroactive andthus can also be detected and measured in accordance with the foregoinginvention including: unsaturated hydrocarbons, azides, triazines andphenothiazines, amino acids, amines and amides, phenols, aromatic OH,quinolines, quinones, imines, olefins, ketones, aldehydes, esters, andolefinic esters, ethers, organometallics, diazo compounds, nitrocompounds, and halogens. The same reagents which are useful fordissolving these organic substances for liquid chromatography generallycan also be used as the reagent in the process of the present invention.Amongst suitable reagents are mentioned: water, lower alcohols, such asmethanol, ethanol and isopropanol, and mixtures thereof. If required astrong inorganic acid such as hydrochloric acid or phosphoric acid, astrong base such as sodium hydroxide, or a salt such as sodium chloridemay be included in the reagent to release the species of interest from acomplex. For example, for analyzing blood samples for the presence ofTylenol, morphine or heroin in accordance with the present invention asuitable reagent comprises methanol/water/phosphoric acid mixturecomprising about 30% methanol, 0.1 to 1% phosphoric acid, and thebalance water. For analyzing blood samples for essential trace elementssuch as zinc, an aqueous solution of calcium acetate buffered to pH 3has been found to be a suitable reagent. A normal saline reagent may beused to measure glucose in blood or serum.

The electrochemical measuring system of the present invention may alsobe advantageously employed for detecting and measuring substances suchas cyanide, halogen, SO₂ and NO_(x) in biological samples, water orsewage. The electrochemical measuring system of the present inventionmay also be adapted for use in monitoring of electroactive substances inchemical process streams. The required electrode potentials areapproximately the same as would be employed in controlled potentialcoulometric stripping of the same organic substances.

The extreme sensitivity of the electrochemical measuring system of thepresent invention permits accurate measurements in picogram region.Thus, the electrochemical measuring system of the present invention maybe advantageously employed for making soil analysis for agriculturalpurposes and may also be used for metal prospecting. In regard to thislatter feature, the process involves measuring soil and/or water samplestaken in a grid pattern in order to zero in on significant deposits ofselected metals. By way of example, to zero in on deposits of relativelyrare metals such as molybdenum, tungsten, vanadium, titanium and uraniumsoil or water samples taken on a grid are extracted with and analyzed ina reagent comprising alcoholic HCl solution such as a 20% solution ofmethanol in HCl. The electrolyte is then charged to the cell, one of theactive electrode segments is set at an electrical potential to oxidizethe metal of interest while another of the active electrode segments isset at an electrolytic potential to oxidize the metal of interest plusother interferring metals. The required electrode potentials areapproximately the same as would be employed in controlled potentialcoulometric analysis of the same metal or metals. Other metals may bemeasured by changing the electrode potentials and/or the reagent. Forexample, for chromium a preferred reagent is alcoholic hydroxidesolution such as 0.8 normal NaOH in methanol. The use of an alcoholicHCl solution as reagent for electrochemical analysis of molybdenum,tungsten, vanadium, titanium and uranium, and the use of an alcoholichydroxide solution as reagent for electrochemical analysis of chromiumare believed novel.

Gaseous samples and/or airborne samples can also be analyzed by bubblingthe gas or air through a suitable reagent to dissolve the substance ofinterest. The electrolyte can then be charged to the electromechanicalcell as above described, and measurements made in accordance with theforegoing.

One skilled in the art will recognize that the invention is susceptibleto modification. Thus, sensing electrode 34 in accordance with thepresent invention has been shown as comparing two active testingelectrode segments, a reference electrode segment and a counterelectrode segment with the electrical potentials on the two activetesting electrode segments being adjusted according to the particularsubstances being detected and measured. One skilled in the art willrecognize, however, that electrode 34 may comprise a large number ofactive testing electrode segments, e.g. electrode 34 may comprise 50 or100 electrically discrete active testing electrode segments, eachsegment being electrically connected to a different electrical potentialto effectively reproduce an entire current voltage curve. For example,the electrode 34 may comprise twelve active testing electrode segmentsat a series of electrical potentials, which may be 20 to 80 millivoltsoffset. Thus, two electrochemically analyze samples which may contain avariety of electroactive substances of interest in which there are knownor suspected interferring substances, it is a simple matter to store thesignal information from each electrode segment and to select out or sortonly those active electrode segments which are at the particularelectrical potentials which produce the desired electrochemicalreactions, derive signals from those electrochemical reactions, and sum(add or subtract) the signals to arrive at the desired measurement. Theselected active electrode segments may be connected in manually by theoperator, e.g. according to printed instructions. Obviously, such anapparatus may also include a plurality of reagents, supplies, reagentvalves, etc. so that a particular reagent may be introduced depending onthe particular substance being detected and measured.

The foregoing apparatus has been described as being run under operatorcontrol; however, the apparatus can be made to operate automatically asfollows: Referring to FIG. 6, a control synchronizer 85 is provided foractuating a pump and valve timing control 86 and also an analog timingcontrol 87. The analog timing control 87 is in the ready position and isactivated for analysis by a start analysis control 88 which appears onthe apparatus as start test button 24.

Optical sensor 68 whose operation is illustrated in FIG. 5 directs asignal to flow sense circuit 19 which in turn sends a signal to pump andvalve timing control 86 and analog timing control 87. Should the flowthrough line 65 be inadequate for complete flushing of cell 27, thesignal from flow sense circuit 90 operates to turn off pump 60 or closevalve 63 or both, and to inactivate analog timer control 87 so that ananalysis cannot be started without resetting the apparatus.

A power supply 91 operated from an A/C power source 92 supplies avoltage through line 93, a negative voltage through line 94, and aground potential through line 95 which are supplied to the cellpotential control 70. The cell potential control 70 can be controlled bypotential set 96.

In a preferred form of automatic controls the apparatus consists of twosections; analog circuitry for converting, conditioning and displayingelectrochemical signals; and reagent handling circuitry for automaticsample handling.

The analysis cycle is controlled by two sequential timers 87. The firsttiming interval (30 seconds) is initiated after the start analysisswitch 88 is depressed. This sequence is used to bring the cell toequilibrium. The second interval (20 seconds) is the concentrationmeasurement. During this time the electrochemical signal is convertedand displayed. In a preferred form the apparatus displays the "countdown" or "count up" digitally during the measurment. Cell referencepotential is controlled by potentiastat circuit 70 and is set by control96. This potential is applied between the reference electrode segment 45and active electrode segment 42. A difference potential is seen betweenactive electrode segment 43 and reference electrode segment 45. Thisdifference potential is set by offset 2 control operating on currentconvertor subtractor 75. The equivalent potential becomes [E_(set) 1-E_(offset) ].

During the measurement inverval the cell currents are fed intocurrent-to-voltage converter circuit 75 and gained controlled bypotentiometers "Gain 1" and "Gain 2". The difference of the resultingvoltages is taken and fed into the accumulator circuit 76 and integratedduring the measurement interval. The integrated voltage then has the"autoblank" value subtracted from it and gained by calibrate circuitry77.

The resultant value is then displayed on the readout 80 in direct unitsof micrograms of iron per 100 ml (μg %) of serum. When the digitaldisplay stops counting the reagent or electrolyte containing the sampleis flushed into container 28 and a new supply of reagent or electrolyteis introduced into the cell from container 29. When the start testindicator 24 lights up again, the apparatus is ready for a next sample.The entire test may take less than one minute, the largest portion ofwhich is the preliminary mixing time.

Reagent or electrolyte can be automatically charged to the cell in anumber of ways. One way is to automatically fill the cell when the unitswitches from the standby to run position; another way is toautomatically fill the cell at the end of each analysis cycle.

Pump and valve timers are set "on" by the control synchronizer 85 from atrigger signal received by the standby control switch 9or the analysiscycle timer. The solenoid valve 63 is used to control reagent flow intothe cell. A pump supplies nominal pressure (e.g. 4 psi) to reagentsupply 62 and a nominal e.g. vacuum (17" Hg) to drain reservoir 69. Thepressure forces clean reagent through the valve into cell 27. Thisincrease in cell volume is taken off through the drain line to the drainreservoir 69. The reagent inlet valve is timed on for a short time, e.g.8 seconds, and the pump is left on for an additional 2 seconds to drainany excess reagent above a set level from the cell.

A flow sensor 68 consisting of optical sensor 56 and flow sense circuit90 monitors the cell drain line 65 during the reagent flushing cycle. Ifthere is no reagent flow or if a low amount of reagent passes throughthe cell, the flow sense circuit 90 will reset the pump and valve timersand thus prevent the start of an analysis. An audio alarm and indicatorlight (light 23) may also be activated at this time. Thus, a new cyclecannot be started until the operator places the instrument in thestandby condition which resets the flow sense circuit 90.

The flow sense circuit 90 comprises an optical sensor (LED 55 andphototransistor assembly 56, FIG. 5) and is placed at the cell drainline. In operation, the output from flow sense circuit 90 changes from alow voltage (line empty) to a higher voltage level (reagent flowing).This level change is sensed and integrated during the first 4 seconds ofthe reagent cycle. If the integrator voltage is below a preset level atthe end of the 4 second interval, instrument lockout is activated.

In the autoblanking operation, when a blank concentration reading istaken and is to be nulled out of future readings, the unit is switchedfrom "run" to "autoblank". The autoblank set switch is depressed,starting a 4 second timer. The binary coded decimal output from thedisplay is latched in the circuit. This BCD number is then convertedfrom a digital to an analog signal.

An analog voltage of correct polarity and magnitude is fed to thecalibration circuitry and subtracted from the concentration analogvoltage resulting in a zero output to the display.

Alternatively, the apparatus may be made to operate automatically, e.g.by means of switching using a microprocessor. In such case, for a knownsubstance, a tape containing instrument instructions would be insertedin the microprocessor, which then selects the reagent to be added to thecell, and the electrode potentials. The results could then be displayedfor usual observation as on a CRT tube or printed out, or the result,may be read into memory for appropriate mathematical manipulation andthen displayed. For an unknown substance, the instrument could beinstructed to connect a plurality of electrically discrete activetesting electrodes at different electrical potentials to thus reproducean entire current voltage curve which can then be compared to currentvoltage curves for known electroactive species. The identification ofthe unknown species can be determined by matching curve shapes while theamount of an electroactive species present in the sample can bedetermined from the area under various sections of the curve for theunknown. More specifically, FIG. 7 illustrates a typical current versuspotential chart obtained in accordance with this invention. In thisgraph, the horizontal axis indicates the difference potential, in volts,of working electrodes at increasingly more positive potentials withrespect to the silver/silver chloride reference electrode. The verticalaxis represents the anodic current, in microamperes, at the indicatedpotential. The waves of the current versus potential curves indicate asharp change in current due to the change in concentration of eachelectroactive species as it reacts in the reagent. Since the potentialat which a particular electroactive species reacts is characteristic ofa particular species in a particular reagent, the electroactive speciespresent in the sample are readily identified. Also, since the presenceof any interferring electroactive species is cancelled out by theelectronics, the areas under the peaks are directly related to the totalamount of and thus to the concentration of each electroactive species inthe sample solution.

A feature and advantage of the present invention is that electrochemicalmeasurements are made of charge transfer reactions substantiallysimultaneously with the occurrance of the reactions. Thuselectrochemical measurements in accordance with the present inventioncan be carried out simultaneously on more than one substance of interestin a sample by application of suitable electrical potentials on thevarious active electrode segments; and through signal sorting. Forexample, a blood sample may be tested simultaneously for lead andchromium.

Various other changes will be obvious to one skilled in the art. Forexample, the active electrode segments have been illustrated ascomprising continuous rings or bands; however, one skilled in the artwill recognize that the active electrode segments may compriseindividual dots or segments, or a series of dots or segments. Moreover,while the electrode preferably comprises a hollow cylindrical, thesimilar advantages may be achieved by shaping the electrode as a hollowcone and by providing a stirrer of mating size and shape. Furthermore,one or more active electrode segments may be added to change theelectrode area at selected voltage potentials so as to reduce or nullout otherwise very large signals from interferring electroactivespecies, or reagent signals in the case of very dilute solutions, andthus increase sensitivity to a particular electrochemical species ofinterest, and to balance signals. Moreover, neither the referenceelectrode nor the counter or power electrode need be mounted as segmentsor electrode 34, but can be separately provided in known manner incontact with the solution being measured. For example, the referenceelectrode and/or the counter electrode may be formed in plug 46. Also,the apparatus may comprise more than one reference electrode and/or morethan one counter electrode. The apparatus could also be adapted tooperate as a flow cell to thus provide a continuous profile of aprocess.

In addition to the foregoing, it will be understood, as shown in FIG. 8,that the electrochemical system may comprise a pair of side-by-side cellassemblies 27A and 27B. Cell assemblies 27A and 27B are similar to cellassembly 27 as above described. In this latter case one of the cellassemblies, e.g. assembly 27A is designed to be the analysis cell whilethe other cell assembly 27B is a blank correction cell. Each activeelectrode segment in cell assembly 27A is paired with a correspondingactive electrode segment in cell assembly 27B at the same potential. Inuse sample containing reagent is injected into cell assembly 27A whilepure reagent is injected into cell assembly 27B. The cell contents arestirred at substantially identical rates, and charge transfer signalsderived as before. The signals from the active cell segments in the twocell assemblies 27A and 27B are summed, e.g. as by subtracting thesignals derived from cell assembly 27B from the signals derived fromcell assembly 27A, with the result that all background signals areessentially nulled. An advantage of employing two similar cellassemblies is that signals resulting from impurities in the reagents arenulled. Also, settling effects resulting from a change of reagent,cleaning cycles, etc., are also nulled. Cell assemblies 27A and 27B maycomprise identical active electrode areas, or one of the cellassemblies, (typically the blank correction cell assembly 27B) may bemade smaller than the analysis cell assembly 27A and the differences inactive electrode areas compensated electrically in known manner. Asmentioned above, the stirring rate in cell assembly 27A should besubstantially identical to the stirring rate in cell assembly 27B. Thesimplest way to assure matching is to mechanically connect the stirringmeans 50A and 50B in the two cell assemblies 27A and 27B to a singlemotor.

Alternatively, the two cell assemblies may be stacked one on top ofanother, e.g. as shown in FIG. 9 at 27C and 27D, and the cell contentsstirred by a stirring means 50C and 50D which are mounted on a commonshaft 51A. Obviously, care must be taken to prevent fluid transportbetween the two cell assemblies 27C and 27D. This can be assured byclose manufacturing tolerances and with sealing means as are well knownto one skilled in the art. As before, grooves 52C and 52D are providedon stirring means 50C and 50D, respectively. If desired, these groovesmay be made to run in opposite directions to one another to minimizefluid transport between cell 27C and 27D.

A particular feature and advantage of the present invention whichresults from the use of an electrode having a plurality of activetesting electrodes at different potentials in accordance with the presetinvention is the elimination of capacitance signals which were inherentin prior art electrochemical measuring in which the potential on anelectrode is changed to obtain a measurement.

Still other features, advantages and objects will be obvious to oneskilled in the art.

We claim:
 1. In serum iron testing, a method of electrochemical testingcompatible with micro sized samples of serum comprising:preparing asubstantially iron-free matrix including a lower aliphatic alcohol andbetween about 51/2 Formal and about 81/2 Formal HCl; introducing apredetermined quantity of serum into said matrix to provide anelectrolytic sample, and adding a measured quantity of said samplecontaining a measured quantity of serum to an electrolytic cell;applying to a first electrode in said cell a potential to measure thequantity of copper and iron in said cell; applying to a second electrodein said cell a potential to measure a different quantity selected fromcopper and iron; obtaining signals corresponding to current flow at eachof said electrodes; and comparing said signals to measure the quantityof iron.
 2. The method of claim 1, wherein the potential applied to saidfirst electrode is between about 0.4 and about 0.5 volts and thepotential applied to said second electrode is between about 0.2 andabout 0.3 volts.
 3. The method of claim 2, wherein the second electrodesignal is subtracted from the first electrode signal.
 4. A compositionfor releasing iron from serum for electrochemical testing comprising asubstantially iron-free mixture of a lower aliphatic alcohol, and HClbetween about 51/2 Formal and about 81/2 Formal.
 5. A compositionaccording to claim 4, wherein said alcohol comprises isopropanol.
 6. Acomposition according to claim 5, wherein said HCl concentration isabout 7 Formal. .[.7. Apparatus for electrochemical testing of serumiron comprising:(a) support means; (b) a cell block positioned on saidsupport means and including an electrolytic cell to contain a quantityof a sample serum; (c) an electrode body in said cell having a pluralityof electrically discrete active testing electrode segments and at leastone reference electrode segment, at least a first said active testingelectrode segment being adapted to detect and measure a first quantitycorresponding to both iron and copper, and at least a second activetesting electrode segment being adapted to detect and measure adifferent quantity selected from iron and copper, whereby the quantityof iron can be determined by comparing the electrochemical measures ofsaid electrode segments; (d) means to apply to said first active testingelectrode segment a potential for measuring both iron and copper fromserum; (e) means to apply to said second active testing electrodesegment a potential for measuring a different quantity selected fromiron and copper; (f) means to compare signals corresponding to electriccurrents to said first and second active testing electrode segments todetermine, by said comparison, the quantity of iron in said sample..]..[.8. The apparatus of claim 7, wherein said means to apply a potentialto said first active testing electrode segment is operative over a rangefrom zero to 1 volt, and wherein said means to apply a potential to saidsecond active testing electrode segment is operative with respect tosaid first electrode over a range between zero and 300 millivolts..]..[.9. The apparatus of claim 7, wherein said means to apply a potentialto said first active testing electrode segment is operative betweenabout 0.4 and about 0.5 volts, and said means to apply a potential tosaid second active testing electrode is operative between about 0.2 andabout 0.3 volts..]. .[.10. The apparatus of claim 9, wherein said meansto apply a potential to said first active testing electrode is operativeat about 460 millivolts, and said means to apply a potential to saidsecond active testing electrode is operative at about 250 millivolts..].11. Apparatus for electrochemical testing of serum iron comprising:(a)support means; (b) a cell block positioned on said support means andincluding an electrolytic cell to contain a quantity of a sample ofserum; (c) an electrode body in said cell having a plurality ofelectrically discrete active electrode segments and at least onereference electrode segment, at least a first of said active testingelectrode segments being adapted to detect and measure both iron andcopper, and at least a second of said active testing electrode segmentsbeing adapted to detect and measure a different quantity selected fromiron and copper, whereby the quantity of iron can be determined bycomparing the electrochemical measures of said active testing electrodesegments; (d) stirring means in said cell; (c) sample input means toreceive a measured quantity of a serum sample for testing; (f) inputmeans to introduce electrolyte liquid to said cell; (g) outlet means todischarge electrolyte from said cell; (h) means to supply electrolyteliquid to said cell and to discharge electrolyte through said outletmeans; (i) means to apply to said first active testing electrode segmenta potential for measuring both iron and copper from serum; (j) means toapply to said second active testing electrode segment a potential formeasuring a different quantity selected from iron and copper; and (k)means to compare signals corresponding to electric currents to saidfirst and second active testing electrodes to determine, by saidcomparison, the quantity of iron in said sample. .[.12. In apparatusaccording to claim 11, means to flush electrolyte through said cell toremove electrolyte therefrom and to replace it with fresh electrolyte,said means comprising conduit means to supply electrolyte to said input,conduit means to receive electrolyte from said outlet, an opticalemitter and an optical detector on opposite sides of one of said conduitmeans, and curved transparent walls in said conduit between said emitterand said detector, whereby radiation from said emitter is more fullyfocused on said detector while electrolyte liquid is in said conduit..]..[.13. Apparatus for electrochemical testing of serum ironcomprising;(a) support means; (b) a cell block positioned on saidsupport means and including an electrolytic cell to contain a quantityof a sample of serum; (c) an electrode body in said cell having aplurality of electrically discrete active electrode segments and atleast one reference electrode segment, at least a first of said activetesting electrode segments being adapted to measure a first chargetransfer for both iron and copper, and at least a second of said activetesting electrode segments being adapted to measure a second chargetransfer for a different quantity selected from iron and copper, wherebythe quantity of iron can be determined by comparing the measures of saidcharge transfers; (d) means to apply to said first active testingelectrode segment a first potential for measuring the charge transfer ofboth iron and copper; (e) means to apply to said second active testingelectrode segment a second potential for measuring the charge transferof a different quantity selected from iron and copper; (f) means tocompare signals corresponding to first and second charge transfers todetermine, by said comparison, the quantity of iron in said sample..]..[.14. The apparatus according to claim 13, including means forsubtracting a signal corresponding to said second charge transfer from asignal corresponding to said first charge transfer..]. .[.15. Theapparatus of claim 13, wherein said means to apply said first potentialis operative at about 460 millivolts, and said means to apply saidsecond potential is operative about 250 millivolts..]. .[.16. Apparatusaccording to claim 7, including means to flush from said cellelectrolyte containing a sample and to replace it with newelectrolyte..]. .[.17. Apparatus according to claim 15, includingdigital display means for displaying the result obtained by subtractingsaid second signal from said first signal..]. .[.18. Apparatus accordingto claim 17 including calibration means for converting said display to adigital readout of micrograms of iron per 100 milliliters of serum..].19. The method of claim 3, and including the step of converting theresult of said signal subtraction to a digital display. .[.20. Anelectrode for electrochemical testing adapted to measure simultaneouslyat least two electrolytic potentials with reference to a referencepotential comprising:a hollow cylindrical electrode body of aninsulating material, open at least at one end; a smooth cylindricalinner surface on said electrode body; at least one reference electrodesegment and at least two active testing electrode segments mounted atthe inner surface of said electrode body and having their respectivesurfaces flush with the inner surface of said body, each of saidsegments being electrically insulated from each other electrode segmentand separated therefrom by said insulating material; said referenceelectrode segment having at least its surface formed of a referencemetal; and means to connect each of said active testing electrodesegments to a different electrical potential..]. .[.21. An electrodeaccording to claim 20, having first, second, third and fourth electrodesegments; said first and second segments being active testing electrodesegments adapted to measure simultaneously two electrolytic potentials;said third segment being a reference electrode segment; and said fourthsegment being adapted to provide an electrolytic current sourcedifferent from the potentials on said first, second and third electrodesegments..]. .[.22. The electrode of claim 21, wherein said referenceelectrode segment comprises silver and said first, second and fourthelectrode segments each comprise carbon..].
 23. In serum iron testing byelectrochemical methods, the steps of:preparing a substantially ironfree matrix comprising between 51/2 and 81/2 Formal HCl in isopropanol;adding to said matrix a measured quantity of a serum sample to betested, thereby releasing serum iron from binding to serumcomponents.Iadd., impressing a first potential for measuring the chargetransfer of iron and copper and deriving a signal therefrom; impressinga second potential for measuring the charge transfer of a differentquantity selected from iron and copper and deriving a signal therefrom;.Iaddend.and separating the charge transfer potentials of iron andcopper .Iadd.by simultaneously measuring the charge transfers at saidtwo different potentials; .Iaddend.and thereafter .[.measuring.]..Iadd.comparing said signals whereby to determine .Iaddend.the quantityof electrolytic current corresponding to iron charge transfer in saidsample .Iadd.by comparing said measured charge transfers..Iaddend..[.24. An electroytic cell for electrochemically testing a sample insolution, said cell comprising:a sensing electrode in the form of ahollow cylindrical body defining an enclosure for holding said solutionfor testing, said body (a) being formed of an electrically insulatingmaterial, and (b) having a substantially smooth cylindrical innersurface; at least two active testing electrode segments mounted at theinner surface of said body, said electrode segments having electricallyactive surfaces which are (a) substantially flush with said smooth innersurface, and (b) electrically insulated from one another by saidinsulating material; a counter electrode having an electrically activesurface mounted in said enclosure; a reference electrode having anelectrically active surface mounted in said enclosure; and means forconnecting (a) said at least two active testing segments to differentelectrical potential, (b) said reference electrode to a referencepotentials, and (c) said counter electrode to yet another potentials..]..[.25. An electrolytic cell according to claim 24 wherein said referenceelectrode comprises one or more electrode segments mounted at the innersurface of said body, each of said reference electrode segments havingan electrically active surface which is (a) substantially flush with theinner surface of said body, and (b) electrically insulated from allother electrode segments on said body..]. .[.26. An electrolytic cellaccording to claim 24 wherein said counter electrode comprises one ormore electrode segments mounted at the inner surface of said body, eachof said counter electrode segments having an electrically active surfacewhich is (a) substantially flush with the inner surface of said body,and (b) being electrically insulated from all other electrode segmentson said body..]. .[.27. An electrode according to claim 24 wherein saidreference electrode comprises silver..]. .[.28. An electrode accordingto any one of claims 24 to 26 wherein said reference electrode comprisessilver, and said active testing electrodes and said counter electrodecomprise carbon..]. .[.29. An electrolytic cell assembly comprisingfirst and second electrolytic cells as defined by claim 24, said firstelectrolytic cell being adapted to hold a sample in a reagent and tomeasure charge transfer reactions of said sample and said reagent, saidsecond electrolytic cell being adapted to hold said reagent alone and tomeasure charge transfer reactions of said reagent alone, said first andsaid second electrolytic cells having corresponding active electrodesegments, and means for pairing electrode segments in said firstelectrolytic cell with corresponding electrode segments in said secondelectrolytic cell at the same potential..]. .[.30. In a method ofelectrochemically analyzing a sample material to identify selectedsubstances therein, the steps of: dissolving said sample in a reagent toform a test solution, charging a quantity of said test solution to anelectrolytic cell in contact with a plurality of active testingelectrodes, simultaneously providing a plurality of electricalpotentials across said solution from said plurality of active testingelectrodes, deriving signals from said active testing electrodescorresponding to charge transfer reactions of materials in saidsolution, and sorting said signals to obtain signals which identify saidselected substances..]. .[.31. Apparatus for electrochemical testing asample in solution to identify selected substances therein, saidapparatus comprising at least one electrolytic cell as defined by claim24, and further comprising means for comparing signals corresponding toelectric current flow at at least two active testing segments todetermine, by said comparison, the quantity of said selected substancesin said sample..].
 32. Apparatus .[.according to claim 31, including.]..Iadd.for electrochemical testing a sample in solution to identifyselected substances therein, said apparatus comprising at least oneelectrolytic cell comprising:a sensing electrode in the form of a hollowcylindrical body defining an enclosure for holding said solution fortesting, said body (a) being formed of an electrically insulatingmaterial, and (b) having a substantially smooth cylindrical innersurface; at least two active testing electrode segments mounted at theinner surface of said body, said electrode segments having electricallyactive surfaces which are (a) substantially flush with said smooth innersurface, and (b) electrically insulated from one another by saidinsulating material; a counter electrode having an electrically activesurface mounted in said enclosure; a reference electrode having anelectrically active surface mounted in said enclosure; means forconnecting (a) said at least two active testing segments to differentelectrical potentials, (b) said reference electrode to a referencepotential, and (c) said counter electrode to yet another potential, saidcell being arranged so that the concentration of each ion species isessentially identical at each testing electrode; and, means forcomparing signals corresponding to charge transfer reactionssimultaneously obtained from each of said active testing electrodes, andmeans for comparing signals corresponding to electric current flow at atleast two active testing segments to determine, by said comparison, thequantity of said selected substances in said sample; said apparatusfurther including: .Iaddend. (a) stirring means mounted in said sensingelectrode; (b) sample input means for introducing a measured quantity ofsample into said sensing electrode; (c) input means for introducingelectrolyte liquid to said sensing electrode; (d) outlet means fordischarging electrolyte from said sensing electrode; and (e) means forsupplying electrolyte to said input means.
 33. Apparatus according to.[.claims 31 or.]. .Iadd.claim .Iaddend.32 for electrochemical testing asample selected from the group consisting of blood and serum, containingboth iron and copper, wherein one of said active electrode segments isadapted to measure a first charge transfer for both iron and copper, andanother of said active electrode segments is adapted to measure a secondcharge transfer for a different quantity selected from iron and copper,and including means for applying to said one active electrode segment afirst potential for measuring the charge transfer of both iron andcopper; means for applying to said another active electrode segment asecond potential for measuring the charge transfer of said differentquantity selected from iron and copper; and means for comparing signalscorresponding to first and second charge transfers to determine, by saidcomparison, the quantity of iron in said sample.
 34. Apparatus accordingto .[.claims 31 or.]. .Iadd.claim .Iaddend.32, and including means forflushing electrolyte through said cell to remove electrolyte therefromand to replace it with fresh electrolyte, .[.said.]. .Iadd.a.Iaddend.means for flushing comprising .Iadd.first and second conduits,.Iaddend.a first conduit for receiving spent electrolyte from said cell,an optical emitter and an optical detector on opposite sides of one ofsaid first and second conduits, and curved transparent walls in said oneof said conduits between said emitter and said detector, wherebyradiation from said emitter is more fully focussed on said detectorwhile electrolyte liquid is in said conduit. .[.35. In a methodaccording to claim 30, the step of applying a potential of between aboutzero and 1 volt to one of said active testing electrodes, and apotential of between about zero and 300 millivolts to another of saidactive testing electrodes..]. .[.36. In a method according to claim 30,wherein said sample is suspected of containing a metal selected from thegroup consisting of molybdenum tungsten, titanium, vanadium, anduranium, the improvement wherein said reagent comprises an alcoholic HClsolution..]. .[.37. In a method according to claim 30, wherein saidsample is suspected of containing chromium, the improvement wherein saidreagent comprises an alcoholic hydroxide solution..]. .[.38. In a methodaccording to claim 30, wherein said sample is selected from the groupconsisting of blood and serum and contains both iron and copper, theimprovement wherein said reagent comprises a substantially iron-freemisture of a lower aliphatic alcohol, and HCl between about 51/2 and81/2 Formal..].
 39. In a method according to claim .[.38.]..Iadd.54.Iaddend., including the steps of introducing a predeterminedquantity of said sample into said mixture to form said test solution,and adding a measured quantity of said test solution to an electrolyticcell;applying to a first active testing electrode in said cell apotential to measure the quantity of copper and iron in said cell;applying to a second active testing electrode in said cell a potentialto measure a different quantity selected from copper and iron; obtainingsignals corresponding to current flow at each of said first and secondactive testing electrodes; and comparing said signals to measure thequantity of iron.
 40. In a method according to claim 39, wherein apotential of between about 0.4 and 0.5 volts is applied to said firstactive testing electrode, a potential of between about 0.2 and 0.3 voltsis applied to said second active testing electrode, and the signal fromsaid second active testing electrode is subtracted from the signal fromsaid first active testing electrode.
 41. In a method according to claim40, wherein said potential applied to said first active testingelectrode is about 460 millivolts and the potential applied to saidsecond active testing electrode is about 250 millivolts.
 42. In a methodaccording to claim .[.38.]. .Iadd.54.Iaddend., wherein said alcoholcomprises isopropanol.
 43. In a method according to claim .[.38.]..Iadd.54.Iaddend., wherein said HCl concentration is about 7 Formal. 44.A composition according to claim 4, and including a minute quantity ofsilver .[.iron.]. .Iadd.ion..Iaddend. .Iadd.45. The method ofelectrochemically analyzing the concentration of a first ion species ina sample which also contains a second ion species comprising the stepsof dissolving said sample in a reagent to form a reagent containing saidtest solution, charging a quantity of said reagent containing said testsolution to an electrolytic cell in contact with a plurality of activetesting electrodes, applying to a first active testing electrode segmenta first potential for measuring the charge transfer of both said ionspecies, applying to a second active testing electrode segment a secondpotential for simultaneously measuring the charge transfer of adifferent quantity selected from one of said ion species whilemaintaining essentially the same concentration of ion species at bothelectrodes, said first active testing electrode segment and said secondactive testing segment being arranged to measure charge transfers onsaid reagent containing said test solution having essentially the sameion concentration in solution at both said electrode segments; andcomparing signals corresponding to first and second charge transfers todetermine, by said comparison, the quantity of said first species insaid sample..Iaddend. .Iadd.46. The process of claim 45 which alsoincludes the electrochemical measuring of the concentration of a secondion species in the presence of a third ion species comprising theadditional steps of applying to a third active testing electrode segmenta third potential for simultaneously measuring the charge transfer of athird quantity selected from one or more of said ion species,andcomparing signals corresponding to first, second and third chargetransfers to determine, by said comparison, the quantity of said firstand second species in said sample..Iaddend. .Iadd.47. The process ofclaim 45 which includes the additional steps of using a pair ofelectrolytic cells, charging to one of said cells said sample-reagentmixture and to the other of said cells reagent free of said sample,applying equivalent potentials to first and second active testingelectrodes in said second cell which are equivalent to the potentialsapplied to the equivalent electrodes in the first cell, and pairingelectrodes in said first electrolytic cell with corresponding electrodesegments in said second electrolytic cell so as to null backgroundsignals due to charge transfer measurements of the reagent and itsimpurities..Iaddend. .Iadd.48. Apparatus for electrochemical measuringof the concentration of a first ion species in a sample dissolved in areagent which also contains a second ion species comprising:(a) asupport arm; (b) a cell block positioned on said support means andincluding an electrolytic cell to contain a quantity of reagentcontaining both said ion species; (c) an electrode body in said cellhaving a plurality of electrically discrete active testing electrodesegments and at least one reference electrode segment, at least a firstof said active testing electrode segments being adapted to measure afirst charge transfer for both said ion species and at least a second ofsaid active testing electrode segments being adapted to simultaneouslymeasure a second charge transfer for a different quantity selected fromone of said ion species, said test electrodes being arranged to measurecharge transfers on a reagent containing said test solution havingessentially the same ion concentration in solution of both testelectrodes, whereby the quantity of said first species can be determinedby comparing the measures of said charge transfers; (d) means to applyto said first active testing electrode segment a first predeterminedpotential for measuring the charge transfer of both said ion species;(e) means to apply to said second active testing electrode segment asecond predetermined potential for simultaneously measuring the chargetransfer of a different quantity selected from one of said ion species;(f) means to compare signals corresponding to first and second chargetransfers to determine, by said comparison, the quantity of said firstspecies in said sample; and (g) means for stirring the reagent adjacentthe electrode segments during measurement..Iaddend. .Iadd.49. Anelectrode system, for electrochemical testing of a solution containingseveral ion species and adapted to measure simultaneously at least twoelectrolytic potentials with reference to a reference potentialcomprising:a hollow cylindrical electrode body of an insulatingmaterial, open at least at one end; a smooth cylindrical inner surfaceon said electrode body; at least one reference electrode segment and atleast two active testing electrode segments mounted at the inner surfaceof said electrode body and having their respective surfaces flush withthe inner surface of said body, each of said segments being electricallyinsulated from each other electrode segment and separated therefrom bysaid insulating material; said reference electrode segment having atleast its surface formed of a reference metal; means to connect each ofsaid active testing electrode segments to a different electricalpotential, said electrode system being arranged so that theconcentration of each ion species is essentially identical at eachtesting electrode; means for generating signals corresponding to chargetransfer reactions simultaneously obtained from each of said activetesting electrodes and for comparing said generated signals; andstirring means for continuously mixing the solution so that theconcentration of each ion species is essentially identical at eachtesting electrode..Iaddend. .Iadd.50. An electrolytic cell forelectrochemically testing a sample containing several ion species insolution, said cell comprising:a sensing electrode in the form of ahollow cylindrical body defining an enclosure for holding said solutionfor testing, said body (a) being formed of an electrically insulatingmaterial, and (b) having a substantially smooth cylindrical innersurface; at least two active testing electrode segments mounted at theinner surface of said body, said electrode segments having electricallyactive surfaces which are (a) substantially flush with said smooth innersurface, and (b) electrically insulated from one another by saidinsulating material; a counter electrode having an electrically activesurface mounted in said enclosure; a reference electrode having anelectrically active surface mounted in said enclosure; means forconnecting (a) said at least two active testing segments to differentelectrical potentials, (a) said reference electrode to a referencepotential, and (c) said counter electrode to yet another potential, saidcell being arranged so that the concentration of each ion species isessentially identical at each testing electrode; means for comparingsignals corresponding to charge transfer reactions simultaneouslyobtained from each of said active testing electrodes; and stirring meansfor continuously mixing the solution so that the concentration of eachion species is essentially identical at each testing electrode..Iaddend..Iadd.51. Apparatus for electrochemical testing of serum ironcomprising:(a) support means; (b) a cell block positioned on saidsupport means and including an electrolytic cell to contain a quantityof a sample of serum; (c) an electrode body in said cell having aplurality of electrically discrete active electrode segments and atleast one reference electrode segment, at least a first of said activetesting electrode segments being adapted to detect and measure both ironand copper, and at least a second of said active testing electrodesegments being adapted to detect and measure a different quantityselected from iron and copper, whereby the quantity of iron can bedetermined by comparing the electrochemical measures of said activetesting electrode segments; (d) stirring means in said cell; (e) sampleinput means to receive a measured quantity of a serum sample fortesting; (f) input means to introduce electrolyte liquid to said cell;(g) outlet means to discharge electrolyte from said cell; (h) means tosupply electrolyte to said cell and to remove electrolyte therefrom andto replace it with fresh electrolyte, said means comprising conduitmeans to supply electrolyte to said input, conduit means to receiveelectrolyte from said outlet, an optical emitter and an optical detectoron opposite sides of one of said conduit means and curved transparentwalls in said conduit between said emitter and said detector, wherebyradiation from said emitter is more fully focussed on said detectorwhile electrolyte liquid is in said conduit; (i) means to apply to saidfirst active testing electrode segment a potential for measuring bothiron and copper from serum; (j) means to apply to said second activetesting electrode segment a potential for measuring a different quantityselected from iron and copper; and (k) means to compare signalscorresponding to electric currents to said first and second activetesting electrodes to determine, by said comparison, the quantity ofiron in said sample..Iaddend. .Iadd.52. In a method of electrochemicallyanalyzing a sample material to identify selected substances therein,wherein said sample is suspected of containing a metal selected from thegroup consisting of molybdenum, tungsten, titanium, vanadium anduranium, the improvement which comprises dissolving said sample in areagent comprising an alcoholic HCl solution to form a test solution,charging a quantity of said test solution to an electrolyte cell wheresaid test solution is in contact with a plurality of differentelectrical potentials across said solution from a plurality of activetesting electrodes, deriving signals from said active testing electrodescorresponding to charge transfer reactions of materials in saidsolution, and sorting said signals to obtain signals which identify saidselected substances..Iaddend. .Iadd.53. In a method of electrochemicallyanalyzing a sample material to identify selected substances therein,wherein said sample is suspected of containing chromium, the improvementwhich comprises dissolving said sample in a reagent comprising analcoholic hydroxide solution to form a test solution, charging aquantity of said test solution to an electrolytic cell in contact with aplurality of active testing electrodes, simultaneously providing aplurality of different electrical potentials across said solution from aplurality of active testing electrodes, deriving signals from saidactive testing electrodes corresponding to charge transfer reactions ofmaterials in said solution, and sorting said signals to obtain signalswhich identify said selected substances..Iaddend. .Iadd.54. In a methodof electrochemically analyzing a sample material to identify selectedsubstances therein, wherein said sample is selected from the groupconsisting of blood and serum and contains both iron and copper, theimprovement which comprises dissolving said sample in a reagentcomprising a substantially iron-free mixture of a lower aliphaticalcohol, and HCl between about 51/2 Formal and 81/2 Formal to form atest solution, charging a quantity of said test solution to anelectrolytic cell where said test solution is in contact with aplurality of different electrical potentials across said solution from aplurality of active testing electrodes, deriving signals from saidactive testing electrodes corresponding to charge transfer reactions ofmaterials in said solution, and sorting said signals to obtain signalswhich identify said selected substances..Iaddend. 93